1. Field of the Invention
The present invention relates to a method of preparing glycoproteins, in particular recombinant glycoproteins, having a high degree of sialylation but a low content of N-glycolylneuraminic acid (Neu5Gc). The method includes the cultivation of cells, in particular host cells, in the presence of (a) dimethyl sulfoxide (DMSO), (b) N-acetylmannosamine (NAcMan), (c) N-acetylglucosamine (NAcGlc), or (d) any combination of two or more thereof. That is, according to the present invention DMSO, NAcMan, and NAcGlc are used as cell culture medium additives.
2. Description of Related Art
Approximately 50% of the proteins produced in eukaryotic cells are glycosylated, which means that they are modified with one ore more oligosaccharide group(s). Usually said glycosylation occurs in two types: O-linked oligosaccharides (attached to serine or threonine residues of the polypeptide backbone) and N-linked oligosaccharides (attached to asparagine residues present within a specific target sequence). The glycosylation profile of these glycoproteins is essential to ensure structural, biological, and clinical stability. Glycosylation of therapeutic proteins plays an essential role in pharmacokinetics, pharmacodistribution, protection from proteolytic degradation, solubility, and receptor binding (Werner et al., 2007). A high degree of terminal sialylation of glycans is important in therapeutic glycoproteins in order to avoid the effect of asialoglycoprotein receptors present in the liver and macrophages, which receptors cause the removal of the glycoproteins from the circulatory system, unless they are sialylated. However, it is desirable that the content of one specific representative (N-glycolylneuraminic acid, Neu5Gc) making up and contributing to sialylation in glycoproteins is low relative to the principal sialic acid (N-acetylneuraminic acid, Neu5Ac) and all other sialic acids (for more details, see below). Thus, there are “good” sialic acids, the principal representative being Neu5Ac, and one “bad” sialic acid: Neu5Gc.
Sialylation is the last intracellular stage of the glycosylation process and involves the enzymatic transfer of sialic acid from a nucleotide sugar precursor, CMP sialic acid, to a galactose moiety available on the emerging glycan structure that is attached to the newly synthesised protein. By the introduction of new or additional N-linked glycosylation sites into the polypeptide backbone, it is possible to create therapeutic glycoproteins that contain additional oligosaccharides which lead to an increased sialic acid content. These proteins show a prolonged serum half-life and an increased biological activity.
Sialic acids compose a family of about 40 derivates of neuraminic acid, which is a nine-carbon atom sugar (e.g., Schauer, 2000). The amino group at position 5 of neuraminic acid is usually acetylated, and this leads to N-acetylneuraminic acid (Neu5Ac), the most widespread form of sialic acids, as mentioned above. A high degree of sialylation of a glycoprotein is thus tantamount to a high degree of Neu5Ac in the glycoprotein and, due to the carboxylic acid function in the neuraminic acid derivatives, also tantamount to an increased acidity of the glycoprotein: the higher the degree of sialylation, the more acidic is the isoform of the glycoprotein.
One of the common modifications of Neu5Ac is the substitution of one of the hydrogen atoms of the acetyl group by a hydroxyl group (—(O═)C—CH3→—(O═)C—CH2OH). The substitution is regulated by the enzyme cytidine-5′-monophosphate-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase) and results in N-glycolylneuraminic acid (Neu5Gc), commonly found in many animal species, but never in normal chicken and human tissues (e.g., Corfield and Schauer, 1982). CMP-Neu5Ac hydroxylase was found to be attached to the surface of the nucleus and to some neighbouring microsomes, and the conversion of Neu5Ac into potentially immunogenic Neu5Gc to take place in the cytosol after CMP-Neu5Ac synthesis in the nucleus.
The catalytic properties including inhibition of CMP-Neu5Ac hydroxylase in vitro have been studied. CMP-Neu5Ac hydroxylase is markedly inhibited by a relatively small increase of the ionic strength. 80% inhibition was obtained with Cu2+, 50% inhibition with Ni3+, Mn2+, and Co2+, and 30% inhibition with Zn2+. Inhibition studies using iron and other metal ligands were also performed obtaining 60% inhibition with the metal ion chelator EDTA. 100% inhibition was accomplished with the iron chelator tiron, 25% inhibition with the iron chelator ferozzine, and 25% inhibition with the zinc chelator phenanthreoline. One scientific paper reported that the only possibility for regulating the proportion of Neu5Gc in the sialic acid pool is to change the activity of CMP-Neu5Ac hydroxylase (Traving and Schauer, 1998).
The immunological implications of Neu5Gc in humans are not fully understood, but it is well known in the art that an increase in the Neu5Gc content of glycoproteins correlates with enhanced antigenicity and, in particular, immunogenicity of the glycoproteins, an undesirable feature for therapeutic proteins. As chickens do not possess Neu5Gc, they were used to shed light on its possible immunogenicity. For example, recombinant human erythropoietin (rhEPO) produced by Chinese hamster ovary (CHO) cells has been reported to contain small amounts (1% of total sialic acids) of Neu5Gc (throughout the entire description below including the examples, any % data for the Neu5Gc content is % (w/w) and refers to the amount of Neu5Gc relative to the total amount of sialic acids in the respective glycoprotein). Though chickens immunised with rhEPO did not produce a significant titre of a respective antibody, a significant titre of antibodies against Neu5Gc was obtained from chickens immunised with (i) fetuin (which has a content of Neu5Gc amounting to 7%) and (ii) GM3 with no Neu5Ac moieties at all (because all Neu5Ac moieties attached to the protein had been converted into Neu5Gc). Therefore, it is readily apparent that the prime objectives for the pharmaceutical industry producing glycoprotein biopharmaceuticals is (i) to keep the content of Neu5Gc as low as possible, preferably below 1%, more preferably even below 0.8%, (ii) to closely monitor the Neu5Gc levels of the glycoprotein biopharmaceutical, and optionally (iii) to reduce the Neu5Gc content of the glycoprotein biopharmaceutical.
Methods and strategies to increase the efficiency of recombinant protein production have been described in the art. Amongst others, butyric acid, glycerol, and dimethyl sulfoxide (DMSO), if added to a CHO cell culture, were found to improve productivity of the protein production process. Simultaneously, these chemicals also induced G0/G1 phase growth arrest and cell growth cessation. Also, DMSO as a cell culture additive (partially) arrests the cells in the G0/G1 phase of the cell cycle, promotes proper protein folding, and enhances protein synthesis.
The literature likewise describes methods to increase the degree of glycosylation and sialylation of recombinant proteins. For example, EP-B 1 543 106 describes a method for increasing the degree of sialylation of glycoproteins produced by mammalian cells. The cell culture medium is supplemented with the key intermediates of the metabolic pathway leading to sialylation of proteins. A combination of galactose (Gal, preferably 0.1 to 40 mM) and fructose (Fru, preferably 1 to 10 mM), with or without mannose (Man, preferably 0.5 to 20 mM, if added) and N-acetylmannosamine (NAcMan, preferably 0.8 to 4 mM, if added) were employed, resulting in an increase of the degree of sialylation.
Another report, Gu and Wang (1998), describes the increase of the intracellular pool of CMP-sialic acid, i.e., the nucleotide sugar substrate for sialylatransferase and, as a consequence thereof, a 15% increase of sialylated interferon-γ was observed.
Yamaguchi et al. (2006) teach that NAcMan is a Neu5Ac (sialic acid) precursor and can be fed to protein-expressing cells to maximise the final sialic acid content of a glycoprotein. In this respect, the authors disclose a large-scale method for NAcMan production from Neu5Ac. Another cell culture process for the production of glycoproteins with increased sialic acid content is set out in EP-B 1 092 037. The experimentators added copper ions to the cell culture in a concentration effective to stabilise the sialic acid content.
A number of enzymes involved in glycosylation (dolichol-linked oligosaccharide synthesis, enzymes that catalyse the addition of O-linked carbohydrates, β-galactoside-α-1,3-galactosyltransferase) utilise divalent cations (e.g. Mn2+) as co-factors. The use of manganese in a concentration of 0.01 μM to 40 μM is described in US-A 2007/0161084 as a method for improving glycosylation and sialylation of glycoproteins.
Finally, U.S. Pat. No. 5,459,031 describes a method for decreasing the Neu5Gc content in proteins by manipulating the culture environment in a way as to increase the concentration of dissolved carbon dioxide and/or carbonate species. The authors managed to control the amount of sialic acid derivates on recombinant glycoproteins and to decrease the Neu5Gc level on the native or recombinant proteins.
As shown above, there is literature available addressing an increase of the degree of sialylation, which means in particular an increase of the Neu5Ac content. However, an increased Neu5Ac content can also lead to an increased Neu5Gc content which is quite undesirable for therapeutic glycoproteins (see above). Only U.S. Pat. No. 5,459,031 describes a method for the production of highly sialylated recombinant glycoproteins with low levels of Neu5Gc. However, according to said patent the content of Neu5Gc is controlled by adjusting and monitoring the level of CO2 in a reaction mixture during protein biosynthesis. Since it is difficult to control the level of CO2 in simple batch and fed batch processes, that method would appear to be limited to repeated batch and perfusion processes.
To summarise the prior art, in particular that described above, there is only limited literature available that is focusing directly on the production of (recombinant) glycoproteins exhibiting a low Neu5Gc content. Accordingly, there is a need for a fast, easy, and cost-effective method that is easily applicable also in a batch and fed batch process to achieve (and maintain) an increased degree of sialylation in (therapeutic) glycoproteins, while the content of (antigenic and thus unwanted) Neu5Gc on said glycoproteins is concomitantly decreased or maintained at low levels.